Effects Of Mesenteric Lymph Drainage Alleviating The Endoplasmic Reticulum Stress In Hemorrhagic Shock Rats

Hemorrhagic shock is a critical condition characterized by insufficient tissue perfusion and oxygen supply. The hypo-perfusion state can trigger endoplasmic reticulum stress and the ensuing structural damage and cell apoptosis, which is considered to be the main factor inducing multiple organ dysfunction or even failure, and ultimately death. In recent years, studies have shown that, in critical conditions, the return of mesenteric lymph to systemic circulation is a pivotal reason of remote organ injury, such as lung, liver, heart and kidney, and using the ligation of mesenteric lymph duct or the drainage of mesenteric lymph blocking the return of lymph to circulation may alleviate organ inflammation, free radical injury and energy metabolism dysfunction. However, it is not known whether the mechanism that the organ protective effects induced by the blocking return of mesenteric lymph to systemic circulation is related to the reduction of endoplasmic reticulum stress (ERS).The present study, observeded the effects of the mesenteric lymph drainage on glucose-regulated protein78(GRP78) and calreticulin (CRT), the molecular markers of endoplasmic reticulum stress, in lung, liver, kidney and myocardium, in a rat model of hemorrhagic shock,. Furthermore, the cell apoptosis and cell apoptosis-related molecules C/EBP homologous protein (CHOP) and cysteinyl aspartate specific proteinase12(caspase-12), were detected in the present study to determine the role of mesenteric lymph drainage playing in the tissue endoplasmic reticulum stress during hemorrhagic shock.Firstly, eighteen male Wistar rats were randomized to sham, shock, shock plus mesenteric lymph drainage (shock+drainage) groups. The operation was performed on femoral region after all the animals were anesthetized with pentobarbital. The right femoral vein was separated and cannulated for anticoagulation with heparin sodium. A minimally heparinized polyethylene catheter was introduced into the right femoral artery used to continuously monitor the animals’mean artery pressure (MAP) throughout the experiment. Another catheter was inserted into the left femoral artery for bleeding by a syringe pump. A laparotomy was carried out to separate the mesenteric lymph duct from surrounding connective tissues. After a30-min stabilization period, blood was withdrawn from the right femoral artery to a MAP of40mmHg within10min at an even speed and was maintained at this level by withdrawing or reperfusion shed blood as needed, thus, the hemorrhagic shock model was established in shock and shock+drainage groups. After90min of hypotension, the ejective blood and the equal Ringer’s solution were reperfused in30min through right femoral vein, the MAP was observed to3h after infusion end. In the shock+drainage group, after infusion finished, the mesenteric lymph duct was cannulated and mesenteric lymph was drained up to3hours after shock. In the sham group, the rats were anesthetized, cannulated and operated as described above, but no blood was withdrawn or infused.The specimens of lung, liver, kidney and myocardium were harvested at3h of hypotension in rats of shock and shock plus drainage group and at corresponding time point in sham group for the next experiments. Part of the animal specimens was prepared10%(V/V) tissue homogenate for ELISA examination of GRP78, CRT, CHOP and Caspase-12. Part of the animal specimens was prepared single-cell suspension for FCM examination of cell cycle rate. Part of the animal specimens was fixed in paraformaldehyde for examination of cell apoptosis using DAPI staining.Results showed that the levels of GRP78and CRT, the molecular markers of ERS, of lung, liver, kidney and myocardium in the shock group were increased significantly when compared with those in the sham group, the GRP78of lung, liver, kidney and myocardium and the CRT levels of lung, liver and kidney in the shock plus drainage group obviously were decreased when compared with those in the shock group and, but still increased compared to those in sham group. Results also showed that the levels of CHOP and Caspase-12, trigger cell apoptotic molecules recruited by ERS, in lung, liver, kidney and myocardium of shock group were enhanced markedly when compared with those of sham group; the CHOP levels of lung, liver, kidney and myocardium and the Caspase-12levels of kidney and myocardium in shock plus drainage group were decreased significantly when compared with those of shock group; and, furthermore, the CHOP levels of lung, liver and myocardium and the Caspase-12levels of lung and liver were still enhanced markedly when compared with those of sham group.Furthermore, the results showed that the cells arrested at Go/G1phase in hepatic and renal tissues in the shock group were remarkably indreased than those of sham group; and the proportion of S and G2/M cell population and proliferation index (PI) in pulmonary, hepatic and renal tissues in shock group were obviously decreased than those of sham group. The mesenteric lymph drainage caused the proportion of G0/G1cells decreased and PI increased. Meanwhile, the ratio of apoptosis in lung, liver, kidney and myocardium in the shock group were signally increased than that of the sham group, so the mesenteric lymph drainage decreased the apotosis of lung, liver, kidney and myocardium tissues.In summary, the ERS is one of pathogenesises responsible for organ injury following hemorrhagic shock. The drainage of mesenteric lymph can attenuate the levels of molecular markers of ERS and trigger molecules of cell apoptosis. The present finding provides an experimental evidence for targeting the return of mesenteric lymph to prevent and treat critical diseases in clinic.